It is well-known that most of the biological processes in animals, including pathological processes, are governed on the cellular level by proteins. Acting directly (e.g. structurally) or through their enzymatic functions, proteins contribute to many pathological processes in animals and humans. While classical therapeutic methods have generally focused on interactions between chemical compounds and proteins, some investigators haw recently focused on treating disease states by modulating the intracellular manufacture of proteins through interactions between putative therapeutic compounds and intracellular genetic material, such as polynucleotides. Such investigators have proven that modulating the production of proteins through interactions with intracellular polynucleotides, such as mRNA, can produce therapeutic results. Polynucleotide-focused drug discovery efforts have produced therapeutic compounds possessing excellent therapeutic activity and minimal undesirable side effects.
One method for specific modulation of gene expression is through the activity of oligonucleotides or oligonucleotide analogs as “antisense” agents. In general, “antisense” methodology involves specific and selective interaction between oligonucleotides or oligonucleotide analogs and complimentary intracellular nucleic acid sequences (e.g. single stranded DNA or mRNA) to modulate transcription or translation of the sequences. In many cases, the interaction between oligonucleotides or oligonucleotide analogs and their intracellular compliments takes place through complimentary base pairing, also known as Watson-Crick hybridization, although other modes of hybridization are also possible.
As oligonucleotides and oligonucleotide analogs have acquired acceptance as promising therapeutic agents, the demand for such compounds has greatly increased. Experimentation has provided three principal methods for the synthesis of oligonucleotides. Reese described the phosphotriester method in Tetrahedron 1978, 34, 3143; Beaucage described the phosphoramidite method in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs; Agrawal, ed., Humana Press: Totowa, 1993, Vol. 20, 33-61; and Froehler described the H-phosphonate method in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs Agrawal, ed.; Humana Press: Totowa, 1993, Vol. 20, 63-80. Of these three synthetic methods, the phosphoramidite method has become the preferred method for synthesizing oligonucleotides on a solid support. The phosphoramidite method includes a step of binding a first 5′-protected nucleoside via the 3′-O to a linker that is in turn conjugated to a solid support. The 5′-protecting group is then removed and a 5′-protected-3′-nucleoside phosphoramidite having a phosphorus protecting group is allowed to react with the support-bound nucleoside, whereby the amine function of the amidite is displaced by the 5′-O of the support-bound nucleoside. The phosphorus, which is in the P(III) oxidation state, can then be oxidized (e.g. sulfurized) to form the P(V) oxidation state. The deprotection, amidite reaction and oxidation steps are repeated until the desired chain length is completed. Once the desired oligonucleotide chain length has been achieved, the phosphorus protecting groups are removed, the oligonucleotide is cleaved from the solid support, and the ultimate 5′-protecting group is cleaved from the oligonucleotide.
In principle, a suitable phosphorus protecting group can be any group that is labile under selective conditions, but that will protect the phosphorus from attack during amidite chain-lengthening, phosphorus oxidation and 5′-deprotection. For instance, Caruthers et al. have taught phosphorus protection using a methyl group (—CH3). See U.S. Pat. Nos. 4,458,066, 4,500,707, 5,132,418, 4,415,732, 4,668,777 and 4,973,679. Caruthers et al. taught removal of the CH3 protecting group (phosphorus-deprotection) by thiophenol in the presence of triethylamine. This procedure for deprotection of methyl-protected phosphoramidites, however, suffers a few notable drawbacks.
One such drawback is that thiophenol is a foul smelling reagent, which is extremely difficult and unpleasant to use. Also, as salts of thiphenol can clog the tubing of automated synthesizers, the user must be careful to maintain reaction conditions within tightly controlled parameters. Thiophenol can also induce methylation of thymine, an undesirable side reaction that alters the structure and the properties of the final oligonucleotide product. Further, removal of methyl groups by this procedure requires reaction periods that can be as long as, or longer than, the time required to synthesize the oligonucleotides. These drawbacks in the removal of alkyl phosphorus-protecting groups have led to the development of alternative approaches to phosphorus-protection and deprotection.
Köster et al. have disclosed a different type of phosphorus protecting group, cyanoethyl, that has gained wide acceptance. See U.S. Pat. Nos. 4,725,677 and Re. 34,069. The cyanoethyl phosphorus protecting group is removed by β-elimination under weakly basic conditions, rather than by direct nucleophilic substitution. While the Köster methodology provides a facile approach to phosphorus deprotection, and is considered an improvement over the Caruthers methodology, it suffers from some drawbacks of its own. For one, cyanoethylphosphoramidites are relatively costly. Also, the free acrylonitrile moiety that arises from β-elimination of the cyanoethyl group can form undesirable adducts. Additionally, acrylonitrile itself is considered toxic, and it would be desirable to reduce, if not eliminate entirely, its production in processes for making pharmaceutical compounds.
There is thus a need for a phosphorus protection/deprotection scheme that would not suffer the drawbacks of using a malodorous deprotecting reagent, such as thiophenol.
There is also a need for a phosphorus protection/deprotection scheme that would provide faster deprotection than dealkylation with thiophenol and triethylamine.
There is also a need for a phosphorus deprotection scheme that would not suffer the drawback of using expensive cyanoethyl protecting groups in the starting materials.
There is also a need for a phosphorus protection/deprotection scheme that would not suffer the drawback of releasing acrylonitrile during deprotection.
There is also a need for such a phosphorus protection/deprotection scheme that can be conveniently carried out using existing automated oligonucleotide synthesizers.
There is thus a need for a method for synthesizing an oligonucleotide that would avoid the disadvantages of using the cyanoethyl group as a phosphorus protecting group during chain elongation.
There is further a need for a non-malodorous reagent capable of removing a phosphorus protecting group from a protected phosphorus during synthesis of oligonucleotides and oligonucleotide analogs.
There is further a need for a method of using a cost effective, non-malodorous reagent for removal of a phosphorus protecting group.